Dyserythropoiesis during anemia related to acute vivax malaria: morphological and transcriptional profile

Background: Anemia is the most frequent complication related to vivax malaria and can be triggered even at low parasitemias. The molecular mechanisms responsible for this clinical syndrome are complex and far from being fully understood. A recent clinical case presenting to our tertiary Hospital demonstrated the presence of Plasmodium vivax (Pv) parasites, dyserythropoiesis and miRNA changes related to erythropoiesis in the bone marrow of an anemic patient (Baro et al., PNTD in press). Here, we aim to follow such observations in a larger study of anemia in acute Pv patients. Materials and Methods: Individuals presenting positive diagnostic for vivax malaria were recruited at Fundação de Medicina Tropical Dr. Heitor Vieira Dourado (Manaus, Amazonas - Brazil). The hemoglobin (Hb) was measured initially by Hemocue® and confirmed by hemocytometer. Male individuals presenting Hb <13.0 g/dL and female individuals presenting Hb <12.0 g/dL were considered anemics. Four mililiters of bone marrow aspirate from illiac crest were obtained at D0 (diagnostic day, previously to treatment) and D42 (convalescence). Erythroid progenitors and reticulocytes were enriched by magnetic-activated cell sorting technology (MACS®) using CD71 microbeads. Morphological analysis of erythropoietic cells was made by light microscopy, counting 500 erythroid progenitors. The enriched cell suspension was stored using Trizol® to posterior total RNA extraction. The integrity of RNA was measured by Agilent® 2100 Bioanalyzer. Eight RNA libraries, corresponding to four D0 and four D42 samples, were generated using Illumina Hi-seq. Results: The morphological analysis showed more abundance of dyserythropoietic cells during acute infection compared to convalescence (p <0.001; unpaired t test). The most frequent dyserythropoiesis signals were: binucleation, irregular shaped nuclei, Howel-jolly bodies presence and megaloblastic cromatin. The RNA libraries showed quality over 85%, presenting around 4.0 x 107 reads of 50 bp and score Phred over 30. Mapping using Homo sapiens GRCh38 genome showed compatibility over 90%. Conclusions: The bone marrow aspirate analysis confirmed the presence of parasites in this central tissue and important morphological alterations in erythroid progenitor cells, probably caused by Pv. Results will be presented on gene ontology and differential expression to determine if genes related to erythropoiesis are dysregulated during acute vivax malaria.